The Human Genome Project and other gene discovery initiatives are dramatically increasing the information available regarding the number, genomic location, and sequences of human genes. Accompanying the expanding base of genetic knowledge are several new technologies geared toward high-throughput mRNA and proteomic analysis of biological samples, allowing a global view of the genes and gene products that reflect normal physiology and pathological states. Utilized together, the expanding genetic database and newly developing analysis technologies hold tremendous potential to increase the understanding of normal cellular physiology and the molecular alterations that underlie disease states. However, many biological specimens, such as whole cell tissue samples, remain uniquely difficult to analyze due to their complex cellular heterogeneity.
The first report of the application of tissue sections directly onto paper strips and subsequent electrophoresis was made by Lindner et al. (1956). Later, Saravis et al. (1979) utilized agarose gels and Bonte (1978) utilized polyacrylamide gels to achieve better separation of the analyzed proteins. As reported in a review by Neuhoff (1980), routine application of these procedures to whole cell tissues was not widespread because of technical difficulties, so methods using extraction of the proteins from the sample through cell lysis before separation predominated.
More recently, Inczedy-Marcsek et al. (1988) described the use of electrophoresis and isoelectric focusing of cryostat samples placed directly upon ultra thin polyacrylamide gels. The use of ultra thin gels allowed for extraction of the proteins from the tissue sample without lysis of the cells of the sample, and did overcome some of the technical difficulties experienced by early workers in this field. Schumacher et al. (1990) also described the use of isoelectric focusing to identify enzymes, glycoproteins, and neuropeptides present in cryostat sections. This process involved the direct placement of the sample upon ultra thin gels, followed by isoelectric focusing. The processes of both Inczedy-Marcsek et al. and Schumacher et al. produce gels in which the proteins or other molecules of interest move through the gel medium according to physical characteristics related to charge and molecular weight. However, these approaches provide information only on the total molecular content of the sample being analyzed, representing the aggregate proteins and nucleic acids present in all of the various cell types present in the specimen.
Isofocusing and electrophoresis processes have been disclosed for cryostat tissue samples, followed by immunochemical analysis. Specifically, Schumacher and Trudrung (1991) and van der Sluis et al. (1988) describe the identification of alkaline phosphatases and peptides such as vassopressin, respectively, through direct tissue isoelectric focusing followed by Western blotting. This immunochemical analysis technique involves the movement of the protein or molecules of interest, through capillary action, from the focusing gel to nitrocellulose membranes. The membrane-bound protein is then detected using immunostaining procedures. Van der Sluis et al. (1988) did attempt to generally localize the proteins within the tissue sample by applying this procedure to a series of sliced tissue sections. However, the immunodetection process was preceded by an isofocusing step, so the results only indicated presence of the protein within a particular tissue sample.
Molecular analysis of cell populations in tissue sections have been performed using immunohistochemistry (IHC) and in-situ hybridization (ISH). The ISH technique is reviewed by Jin and Lloyd (1997), and the IHC technique is reviewed by Grogan (1992). While these techniques have been valuable tools to investigate the cellular localization of a particular protein or mRNA in a complex tissue section, they both suffer from three major drawbacks. First, IHC and ISH are limited to analysis of a single molecular species per sample. Second, artifact staining based on cross-hybridization severely affects the accuracy of the test results. Finally, these methods have limited ability to visualize proteins and mRNAs expressed at moderate or low levels of abundance.
Techniques have been disclosed for separating particular subsets of cells from a whole tissue sample. For example, Emmert-Buck et al. (1996) describe the use of laser-based microdissection techniques to rapidly procure microscopic, histopathologically defined cell populations. Alternatively, tissue arrays, such as those described by Kononen et al. (1998) permit individual molecules to be studied simultaneously in hundreds of separate tissue samples. However, there remains a need in the art for an improved method of analyzing proteins or other molecules of interest present in cellular specimens where the method is capable in some embodiments of providing information concerning the location of the proteins or molecules of interest in the initial tissue sample, and/or provide a method that avoids some of the problems encountered with IHC and ISH.